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PreprintCC BYChemRxiv

Capacitance-based measurement of calcium-mediated vesicle fusion upon microinjection in droplet interface bilayers

  • McKayla Torbett-Dougherty1,2
  • Thomas Shaw3,2
  • Beliz Utebay4,5
  • Vivian Hu1,5
  • Raman Dhiman6,7
  • Kyler Grogan6,7
  • Malika Rao8,7
  • Jonathan Rivnay4,5,1
  • Neha Kamat1,5
  • Manish Kumar6,7,8
  • Stephen A. Sarles1,2,3
8 affiliations
  1. 1Department of Biomedical Engineering
  2. 2University of Tennessee
  3. 3Department of Mechanical and Aerospace Engineering
  4. 4Department of Materials Science and Engineering
  5. 5Northwestern University
  6. 6McKetta Department of Chemical Engineering
  7. 7University of Texas
  8. 8Maseeh Department of Civil, Architectural and Environmental Engineering
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Abstract

The droplet interface bilayer (DIB) offers a tunable and stable model membrane platform for studying transmembrane proteins (TMPs). Yet, the library of TMPs known to incorporate into DIBs remains limited to those that spontaneously self-insert from aqueous microdroplets. Moreover, incorporating TMP-decorated liposomes into DIB droplets prior to bilayer formation can disrupt lipid assembly and leave TMPs poorly protected from the oil-water interface. We overcome these hurdles by leveraging calcium-mediated vesicle fusion (CMVF) to deliver bilayer-stable TMPs into DIBs via post-bilayer formation microinjection. Departing from prior works, we employed capacitance-based measurements of intrinsic membrane potential to quantify fusion-induced changes in DIB lipid composition. Our results show that CMVF of symmetric, fusogenic liposomes with an asymmetric DIB reduces its transmembrane potential by an amount that corresponds to the fused area of the bilayer: injecting ~40 nL of fusogenic liposomes into one 400 nL droplet resulted in ~15% of the bilayer area becoming less asymmetric in leaflet composition. Moreover, repeated injections produced cumulative vesicle fusion, while omission of fusogenic lipids or Ca2+ ions abolished changes in membrane potential. After quantifying the CMVF process, we used it to enable and enhance the incorporation of multiple TMPs into DIBs. First, injection of proteoliposomes containing TREK-1 proteins, which do not readily insert into DIBs without fusion, consistently produced stochastic, multi-channel gating within minutes. TREK-1 channels exhibited bipolar voltage dependence and pharmacological activation by arachidonic acid, confirming native channel function. Separately, injection of proteoliposomes decorated with OmpF proteins yielded comparable increases in nominal membrane conductance to spontaneously-inserted OmpF proteins, but at half the driving voltage and despite delivering 10X less protein, indicating that fusion incorporates functional TMPs more efficiently than diffusion-limited protein insertion. Together, these results establish capacitance-based measurements and CMVF as a quantitatively verifiable route for improving TMP incorporation into planar lipid bilayers.

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Cite

BibTeXRIS
BibTeX
@misc{torbettdougherty2026capacitancebased,
  title = {{Capacitance-based measurement of calcium-mediated vesicle fusion upon microinjection in droplet interface bilayers}},
  author = {McKayla Torbett-Dougherty and Thomas Shaw and Beliz Utebay and Vivian Hu and Raman Dhiman and Kyler Grogan and Malika Rao and Jonathan Rivnay and Neha Kamat and Manish Kumar and Stephen A. Sarles},
  howpublished = {ChemRxiv},
  year = {2026},
  month = aug,
  doi = {10.26434/chemrxiv.15008036/v1},
  url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008036/v1},
  note = {CC BY},
}
RIS
TY  - UNPB
TI  - Capacitance-based measurement of calcium-mediated vesicle fusion upon microinjection in droplet interface bilayers
AU  - McKayla Torbett-Dougherty
AU  - Thomas Shaw
AU  - Beliz Utebay
AU  - Vivian Hu
AU  - Raman Dhiman
AU  - Kyler Grogan
AU  - Malika Rao
AU  - Jonathan Rivnay
AU  - Neha Kamat
AU  - Manish Kumar
AU  - Stephen A. Sarles
PY  - 2026
DA  - 2026/08/30
PB  - ChemRxiv
DO  - 10.26434/chemrxiv.15008036/v1
UR  - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15008036/v1
AB  - The droplet interface bilayer (DIB) offers a tunable and stable model membrane platform for studying transmembrane proteins (TMPs). Yet, the library of TMPs known to incorporate into DIBs remains limited to those that spontaneously self-insert from aqueous microdroplets. Moreover, incorporating TMP-decorated liposomes into DIB droplets prior to bilayer formation can disrupt lipid assembly and leave TMPs poorly protected from the oil-water interface. We overcome these hurdles by leveraging calcium-mediated vesicle fusion (CMVF) to deliver bilayer-stable TMPs into DIBs via post-bilayer formation microinjection. Departing from prior works, we employed capacitance-based measurements of intrinsic membrane potential to quantify fusion-induced changes in DIB lipid composition. Our results show that CMVF of symmetric, fusogenic liposomes with an asymmetric DIB reduces its transmembrane potential by an amount that corresponds to the fused area of the bilayer: injecting ~40 nL of fusogenic liposomes into one 400 nL droplet resulted in ~15% of the bilayer area becoming less asymmetric in leaflet composition. Moreover, repeated injections produced cumulative vesicle fusion, while omission of fusogenic lipids or Ca2+ ions abolished changes in membrane potential. After quantifying the CMVF process, we used it to enable and enhance the incorporation of multiple TMPs into DIBs. First, injection of proteoliposomes containing TREK-1 proteins, which do not readily insert into DIBs without fusion, consistently produced stochastic, multi-channel gating within minutes. TREK-1 channels exhibited bipolar voltage dependence and pharmacological activation by arachidonic acid, confirming native channel function. Separately, injection of proteoliposomes decorated with OmpF proteins yielded comparable increases in nominal membrane conductance to spontaneously-inserted OmpF proteins, but at half the driving voltage and despite delivering 10X less protein, indicating that fusion incorporates functional TMPs more efficiently than diffusion-limited protein insertion. Together, these results establish capacitance-based measurements and CMVF as a quantitatively verifiable route for improving TMP incorporation into planar lipid bilayers.
ER  -